ELISA protocol for rapid screening of potential antitubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease - a drug target supported by axenic culture of Mycobacterium tuberculosis H37 Ra strain in the presence of inhibitor.

Background: Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. Aim: To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. Material and Methods: Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. Results: Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5μg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4μg/well, while orlistat showed inhibition of 60% at 0.5μg/well. Inhibition of Mtb H37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4μg/well and orlistat at 0.5μg/well were observed respectively on bacterial growth. Conclusion: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.

Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme

Colonic proteinase: increased activity in patients in patients with ulcerative colitis

In order to study the colonic intraluminal proteinase-antiproteinase imbalance under inflammatory conditions, we determined proteolytic activity (PA), alpha-1-antitrypsin and the activities of trypsin, chymotrypsin and neutrophil elastase in feces from patients with ulcerative colitis (UC) comparing the results with a control group. A fecal sample was obtained from each of 25 patients with ulcerative colitis and 10 control subjects were studied. The severity of the disease was assessed by the Truelove index. Proteolytic activity was measured lesing azocasein as proteolytic substrate. The fecal concentration of alpha-1-antitrypsin was measured by radial immunodiffusion and the activities of the enzymes were measured using specific substrates. We found an increase in fecal PA, alpha-1-antitrypsin and neutrophil elastase in patients with UC and the correlation between the severity of the disease and the PA was statistically significant (r = 0.62, P < 0.05). We conclude that elevated colonic proteinase activity could contribute to the pathophysiology of ulcerative colitis.

Heart prolyl endopeptidase activity in one-kidney, one clip hypertensive rats

1. Heart mass, prolyl endopeptidase activity and fractionated proteins from heart tissue were studied in one-kidney, one clip hypertensive rats (N=6) and compared to sham-operated rats (N=6). 2. Body weigh, arterial pressure and tissue mass were measured 4 weeks after artery clipping Z-Gly-Pro-p-nitroaniline hydrolysis was used to measure tissue prolyl endopeptidase activity in the homogenate. Protein was fractionated into the soluble and myofibrillar fractions. 3. In the normotensive rats, prolyl endopeptidase activity expressed in terms of protein specific activity (µM substrate hydrolyzed h-1 mg supernatant protein-1) occurred in atria and was 2.5-fold higher than in the ventricles (3.79 ñ 0.20 vs 1.44 ñ 0.02, P<0.05). In the one-kidney, one clip hypertensive rats, the left ventricle tissue increased 1.7-fold (2.27 ñ 0.11 vs 3.72 ñ 0.11 mg wet weight tissue/g body weight, P<0.001), the soluble protein fraction (54.86 ñ 3.60 vs 57.38 ñ 6.64 mg/g wet weight tissue) was unchanged, while the myofibrillar fraction increased 1.9-fold (118.9 ñ 9.09 vs 229.8 ñ 8.47 mg/g wet weight tissue, P<0.001). 4 The specific activity of the atrial and ventricular prolyl endopeptidase decreased in atria and increased in ventricles as the result of hypertension (3.79 ñ 0.2 vs 2.84 ñ 0.13 and 1.44 ñ 0.02 vs 1.87 ñ 0.13; respectively). These regional differences in prolyl endopeptidase enxyme content caused by one-kidney, one clip hypertension in neurosecretory and non-neurosecretory heart areas suggest that this enzyme plays a local role in the turnover of specific polypeptides

Malaria parasites: enzymes involved in red blood cell invasion

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a performed serine-protease active site but not detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erytrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented